Siderophore uptake experiments

Siderophore-mediated iron uptake was performed as described by Fuchs et al. (2001) and adapted by Hoegy and Schalk (2014). Briefly, labeled ferrisiderophores were prepared by mixing a 4μl⁵⁵FeCl₃ 250 μM (Amersham) with 4 μl of a 1 mM pyoverdine solution obtained from the cultures of the different strains. After 30 min of incubation at room temperature, this mixture was diluted with Tris-HCl pH 8 (50 mM) to obtain a 10 μM siderophore-⁵⁵Fe complex.

Pseudomonas strains to be used as siderophore uptaking cells were grown in CAA medium for 24 h at 30°C. Then, bacterial cells were washed twice in CAA medium to remove the native siderophore and were re-suspended in Tris-HCl pH 8 (50 mM) to reach an optical density of 1 at 600 nm.

Five microlitres of siderophore-⁵⁵Fe complex were mixed with 500 μl of the cell suspension and incubated for 30 min at 30°C. Then, 100 μl of the labeled bacterial cells were withdrawn and rapidly filtered through 0.45 μm membranes (Whatmann GFB filters). The cells remaining on the filters were thoroughly washed twice with Tris-HCl pH 8 (50 mM) and the radioactivity was measured to determine the amount of labeled iron incorporated during the incubation time Gamma emission was counted using a PerkinElmer gamma counter. Iron uptake of heterologous pyoverdine was expressed in percentage terms compared with the homologous pyoverdine uptake.